Engineering Enhanced Function into G. dibranchiata Monomer Hemoglobins

本提案的长期目标是了解血红素蛋白结构如何调节血红素蛋白的反应性。该项目以双分枝甘油单体血红蛋白为重点。有三种不同的单体血红蛋白，其中两种表现出与脊椎动物肌红蛋白相似的结构。尽管如此，它们的动力学性质与肌红蛋白有很大的不同，可能的原因是在每个单体血红蛋白中，远端组氨酸被亮氨酸取代。例如，在氰化物结合的情况下，与脊椎动物的肌红蛋白相比，这些蛋白质明显没有反应性。其中一个珠蛋白cDNA序列已被克隆、测序并在大肠杆菌表达系统中表达。位点导向突变体也被构建，以观察是否可以将增强的反应性构建到自然不反应的结构中。野生型蛋白和位点定向突变体将使用动力学，平衡和核磁共振来实现结构与功能相关的目标。

The long-term objective of this proposal is to understand how heme protein architecture regulates heme protein reactivity. The project has as its focus the Glycera dibranchiata monomer hemoglobins. There are three distinct monomer hemoglobins two of which exhibit structures similar to vertebrate myoglobins. Despite this their kinetic properties differ significantly from myoglobin, for the probable reason that in each of the monomer hemoglobins the distal histidine is replaced by leucine. In the case of cyanide binding, for example, these proteins are notably unreactive in comparison to vertebrate myoglobins. One of these globin cDNA sequences has been cloned, sequenced and expressed in an E. coli expression system. Site-directed mutants have also been constructed to see if enhanced reactivity can be built in to a naturally unreactive structure. Wild-type proteins and site directed mutants will be studied using kinetics, equilibrium and NMR to fulfill the goal of relating structure to function.